It is crucial to comprehend how polylactic acid PLA responds to cyclic loading to guarantee the reliability and durability of 3D-printed parts. Researchers can improve performance in certain applications by adjusting printing conditions, material composition, and post-processing processes by analyzing its fatigue behavior. The purpose of this study is to examine the fatigue behavior of a polymer produced using additive manufacturing. To manufacture the fatigue samples, a fused deposition modeling (FDM) printer was used to 3D print the polylactic acid (PLA) biodegradable polymer. Three different printing orientations were selected: vertical, horizontal [0/90], and horizontal [+45/−45]. Rotary bending fatigue testing was used to assess the fatigue behavior of these samples at different alternate peak bending stress levels, specifically 20, 40, 59, and 79 MPa. The results showed that PLA printed in the horizontal [+45/−45] printing orientation was more fatigue resistant than PLA printed in any other orientation, especially when low load was applied (Nf[+45/−45] = 605700 >> Nf[0/90] = 240600 > Nfvertical = 164225 cycles). Additionally, there is a good agreement between the experimental data and the predicted S-N curves model computed according to Basquin’s initial model of S-N one. Furthermore, at the lowest alternate peak bending stress (20MPa), the fatigue strength of the 3D-printed samples was lower in the vertical direction than it was in the horizontal direction.